Multiplexed tandem mass spectrometry

ABSTRACT

One or more methods for determination and/or deconvolution of complex mass spectra, using quadrupole ion trap mass spectrometry

This application is a continuation in part of and claims prioritybenefit of U.S. application Ser. No. 11/136,127 filed May 24, 2005, andissued as U.S. Pat. No. 7,141,784 on Nov. 28, 2006, and 60/573,980,filed May 24, 2004, each of which is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Quadrupole ion trap mass spectrometers (QITMS) are used to provide rapidand sensitive analyses of a wide range of chemical and biochemicalcompounds. QITMSs and related spectrometric methods are known in the artand are as provided in U.S. Pat. No. 4,540,884, the entirety of which isincorporated herein by reference. Such instruments have begun to play aparticularly important role in proteomics research as their favorablecharacteristics are applied to the identification, quantitation, andstructural elucidation of peptides and proteins. One limitation with theQITMS, however, is that the structural analyses provided by thisinstrument is performed in a serial manner in the context of tandem massspectrometry (MS/MS) experiments. With the emerging importance ofproteomics the need for more rapid analyses are being realized.

A range of ions with different mass-to-charge (m/z) values can betrapped simultaneously in a quadrupole ion trap by the application of aradio frequency (rf) voltage to the ring electrode of the device. Thetrapped ions all oscillate at frequencies that are dependent on theirm/z, and these frequencies can be readily calculated. MS/MS is thenperformed by carrying out three steps. First, the analyte ions havingthe single m/z of interest (parent ions) are isolated by changing the rfvoltage applied to the ring electrode and by applying waveforms (i.e.appropriate ac voltages to the endcap electrodes) with the appropriatefrequencies that resonantly eject all the ions but the m/z of interest.Second, the isolated parent ions are then resonantly excited via theapplication of another waveform that corresponds to the oscillationfrequency of the parent ions. In this way, the parent ions' kineticenergies are increased, and they undergo energetic collisions with thebackground gas (helium), which ultimately result in their dissociationinto product ions. Third, these product ions are then detected with theusual mass analysis techniques in QITMS. It is the mass differencesbetween these product ions and their incipient parent ions that providesthe structural information during this MS/MS experiment. This method ofperforming MS/MS is the current state-of-the art in commercial QITMS,and referred to as serial MS/MS.

Multiplexed MS/MS refers to performing MS/MS on ions of multiple m/zratios simultaneously. A primary concern, however, is that uponisolation and dissociation of several compounds simultaneously, theproduct ions that are formed need to be associated with the correctparent ions in order for structural information to be gathered for eachparent ion. During serial MS/MS this is accomplished by isolating anddissociating only one parent ion at a time so that the resultingproducts necessarily come from that parent ion. When one isolates anddissociates multiple parent ions all at once, the normal manner ofrelating which product ions dissociate from each parent ion is lost.

Several protocols for multiplexed MS/MS on Fourier Transform IonCyclotron Resonance (FTICR) mass spectrometers have been reported.Comprehensive 2-dimensional (2-D) methods analogous to 2-D NMR were usedto simultaneously dissociate a collection of parent ions. Attributingthe resulting product ions to the appropriate parent ions relies on asinusoidal pattern of excitation waveforms that produces a modulation inthe product ion abundances that can be later deconvoluted. Hadamardtransform methods have also been used, but like the comprehensive 2-Dapproach multiple spectra are acquired in which different subsets ofparent ions are simultaneously dissociated. The drawback to both theHadamard method and the comprehensive 2-D approach is that these methodsprovide little to no timesavings as compared to the analogous serialapproaches. Further, parent ion dissociations and product ion abundancesdo not always vary in the expected manner. Encoding is dependent uponknown changes in parent ion kinetic energy, but product ion abundancesdo not necessarily change in a direct manner as parent ion kineticenergies (and, thus, collision energies) are changed. The net result isthat product ions may be associated with incorrect parent ions.

Another approach developed recently allows product ion spectra to beobtained from multiple parent ions in a single mass spectrum, whichsignificantly enhances the throughput. Because MS/MS analyses on FTICRmass spectrometers are inherently slower than MS/MS analyses on QITMS,this method is noticeably slower. Furthermore, this approach relies onthe high mass accuracy of the FTICR to identify product ions fromdifferent parent ions by exact mass and database searching.Consequently, this method necessitates the high performance capabilitiesoffered only by FTICR spectrometers, and therefore is not suitable forcheaper and more widely accessible mass spectrometers like QITMS.Further, this method depends upon the compound of interest present in anaccessible data base and effective search capabilities—without which theanalysis is unworkable.

Another QITMS limitation relates to the fact that externally-generatedions of different mass-to-charge (m/z) ratios are trapped with unequalefficiency after being transferred from the ion source. This unequaltrapping efficiency results in mass spectra that do not accuratelyreflect the relative quantities of the analytes in a given sample. Thism/z (or mass) bias arises because there exists, for each m/z ratio witha given kinetic energy, an optimum rf amplitude on the ion trapelectrodes for efficient trapping. Furthermore, this optimum rfamplitude is different for each m/z ion. Such issues arise within oroutside the context of MS/MS analyses.

To overcome mass bias, three different approaches have been used in theart. First, the rf amplitude can be increased in three different stepsduring ion accumulation. This step-wise increase results in optimumtrapping of three different m/z ratios whether or not they exist in thesample. In effect this reduces the severity of the mass bias for alarger range of m/z ions, but does not totally eliminate it; thetrapping efficiency for any give m/z ratio can still vary significantly.The step-wise increase in the rf amplitude cannot provide uniformtrapping efficiency over a wide m/z range. The second approach,described in U.S. Pat. No. 5,729,014, involves a linear increase in therf amplitude during ion injection. This linear increase in amplitude ismeant to achieve, for a very brief time, the optimum rf amplitude foreach ion in a given m/z range. While effective at reducing the mass biasassociated with externally-injected ions, this method raises at leasttwo issues—(1) increasing the rf amplitude in such a manner can lead toion dissociation and thus loss of ion signal and sample integrity; (2)the linear rf amplitude increase (or multiple segments of this linearfunction) only approximates the non-linear increase necessary to matchthe ideally-predicted relationship.

A third approach has been developed in which the rf frequency is changedin the theoretically correct way. U.S. Pat. No. 6,121,610 describes asystem in which the optimum rf frequency is varied in inverse proportionto the square root of the m/z ratio (i.e. (m/z)^(−1/2)). Alternatively,there is described a method in which the rf amplitude is decreasedduring ion injection to accomplish an effect similar to that describedby in the '014 Patent. The methods of the '610 Patent are theoreticallypreferred, but changing the rf frequency is very challenging from anelectronics standpoint. Furthermore, ion losses for low m/z ions canstill readily occur.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C: (A) Mass spectrum of a collection of peptide ions. (B)Spectrum obtained after simultaneously isolating 4 parent ions. (C)Spectrum obtained after simultaneously dissociating the 4 parent ions

FIGS. 2A-B: (A) Spectrum obtained after simultaneously isolating 4parent ions under different accumulation conditions. (B) Spectrumobtained after simultaneously dissociating 4 parent ions in A.

FIGS. 3A-B: (A) Normalized ion abundances as a function of the rfvoltage during ion accumulation. (B) rf voltage required for optimum ionaccumulation as a function of ion m/z ratio.

FIGS. 4A-B: Mass spectra of cytochrome c at LMCO values of (A) 41 and(B) 51.

FIGS. 5A-B: (A) Normalized ion intensity distribution for the +15 chargestate of cytochrome c as a function of LMCO during ion accumulation. (B)Normalized ion intensity distribution for the +14, +15, +17, and +20charge states of cytochrome c as a function of LMCO during ionaccumulation.

FIG. 6: Plot of the optimum LMCO during ion accumulation as a functionof peptide ion m/z.

FIG. 7: Plot of the width of the Gaussian distribution (in LMCO units[Da]) as a function of peptide ion m/z. The average width of 8 Da isshown by the line through the data.

FIG. 8: Illustration of how intensity encoding is accomplished.Acquisition of the primary spectrum (1°) is carried out at the LMCOvalue shown by the solid line, and acquisition of the secondary spectrum(2°) is carried out at the LMCO value shown by the dotted line. The ionintensity change is then predicted using equation 1.

FIGS. 9A-B: (A) Mass spectrum of a mixture of 5 peptides (TRH-Gly,angiotensinogen, leucine enkephalin, β-casomorphin, and leucokinin)acquired at a LMCO value of 48 Da. (B) Mass spectrum of same mixture ofpeptide ions after SWIFT isolation at a LMCO value of 48 Da.

FIG. 10: Product ion spectrum (MS/MS) after simultaneous dissociation of5 peptide ions from FIG. 9B (LMCO accumulation value=48 Da).

FIG. 11: Mass spectrum of a mixture of 5 peptides (TRH-Gly,angiotensinogen, leucine enkephalin, μ-casomorphin, and leucokinin)after SWIFT isolation at a LMCO value of 58 Da.

FIG. 12: Product ion spectrum (MS/MS) after simultaneous dissociation of5 peptide ions from FIG. 11 (LMCO accumulation value=58 Da).

FIG. 13: Ratio spectrum obtained by dividing the primary spectrum (FIG.10) by the secondary spectrum (FIG. 12).

FIG. 14: Expanded region between m/z 450 and 625 of the Ratio Spectrumin FIG. 13.

FIG. 15: Product ion spectrum (MS/MS) of an authentic sample ofleucokinin, which demonstrates the number of its product ions identifiedby the multiplexed MS/MS approach. The peaks with an asterisk (*) abovethem are the product ions that were correctly identified during themultiplexed MS/MS experiment. The peaks with the plus sign (+) abovethem are product ions that were not identified during the multiplexedMS/MS experiment because these ions have m/z values close to those ofother parent ions and thus their detection is precluded by the SWIFTwaveforms used to dissociate these parent ions. The peak with a questionmark (?) above it corresponds to a product ion that was present in theratio spectrum, but its intensity ratio was deemed to be too far fromthe expected intensity ratio for leucokinin (i.e. 1.2).

FIG. 16: Ion intensity distribution for TRH-Gly (m/z 421) as a functionof LMCO during ion accumulation. The solid line shows the LMCOaccumulation value used during acquisition of the spectrum in FIG. 9B,and the dotted line shows the LMCO value used during acquisition of thespectrum in FIG. 11.

FIG. 17: Normalized ion abundance distribution for the protonatedpeptide ion (M+H)⁺ of leucine enkephalin as a function of LMCO duringion accumulation for two different octopole dc settings.

FIG. 18. Ion intensity of representative m/z 556 and 1060 as a functionof low m/z cut off (LMCO), demonstrating trapping efficiency change as afunction of LMCO.

FIG. 19. Ion intensity distributions for m/z 556 at different octopole 1voltages. The octopole 1 voltage is directly proportional to ion kineticenergy during ion injection.

FIG. 20. Ion intensity of m/z 556 and 1060 as a function of low m/z cutoff (LMCO) when ions are injected into the ion trap with higher kineticenergy than in FIG. 18.

FIG. 21. Ion intensity of m/z 556 as a function of low m/z cut off(LMCO) when ions are injected at various kinetic energies.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide one or more multiplexed tandem mass spectrometric methods,thereby overcoming various deficiencies and shortcomings of the priorart, including those outlined above. It will be understood by thoseskilled in the art that one or more aspects of this invention can meetcertain objectives, while one or more other aspects can meet certainother objectives. Each objective may not apply equally, in all itsrespects, to every aspect of this invention. As such, the followingobjects can be viewed in the alternative with respect to any one aspectof this invention.

It is an object of the present invention to provide a methodology forencoding, simultaneously, product ions originating from multiple parentions.

It is another object of the present invention to provide one or moremultiplexed tandem mass spectrometric methods more efficiently from botha cost and time perspective, as compared to analogous serial methods ofthe prior art.

It is another object of the present invention to provide a method fordirect encoding of multiple parent ion abundances, thereby avoiding theinherent non-linearity between collision energies and product ionabundances of the sort which accompany the two-dimensional methods ofthe prior art.

It is a further object of the present invention to provide multiplexedor multiplexed mass spectrometric methods without reliance on data basecomparison or search capabilities.

It can be another object of the present invention, alone or inconjunction with one or more of the preceding objectives, to provide amethod to reduce mass-related bias associated with ion accumulation,such that a resulting mass spectrum can more accurately reflectcomponent species in an analyzed mixture.

It can be another object of the present invention to reduce such biaswith variation in ion kinetic energy, regardless of any one particularquadrupole ion trap mass spectrometer employed.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofvarious embodiments, and will be readily apparent to those skilled inthe art having knowledge of mass spectrometric methods and analyses.Such objects, features, benefits and advantages will be apparent fromthe above as taken in conjunction with the accompanied examples, data,figures and all reasonable inferences to be drawn therefrom, alone orwith consideration of the references incorporated herein.

Accordingly, the present invention can be directed to a method formultiplexed tandem mass spectrometry. Such a method comprises providingfirst spectra comprising a spectrum of a plurality of compound parentions, and a spectrum of at least one product ion of each parent ion, thefirst spectra acquired under a first set of ion trapping conditions;providing second spectra comprising a spectrum of a plurality ofcompound parent ions, and a spectrum of at least one product ion of eachparent ion, the second spectra acquired under a second set of iontrapping conditions; determining the fractional change in each parention accumulated over the first and second spectra; and applying thefractional change to at least one of the product ions in the first andsecond product ion spectra, to determine product ions for each parention.

Trapping conditions can be selected from conditions which would be knownto those skilled in the art made aware of this invention, and caninclude conditions selected from at least one of rf voltage applied to aring electrode during accumulation, and a predetermined low m/z cut off.The spectra can be acquired over different applied rf voltages, or suchcut off values, as expressed by relationships defined more fully, below.Depending upon factors including the number of compounds to be resolvedor the degree of resolution required, such a method can further compriseproviding third spectra comprising a spectrum of a plurality of compoundparent ions, and a spectrum of at least one product ion for each parention, the third spectra acquired under a third set of trappingconditions. Regardless, such a method can further comprise use of aliquid chromatographic separation either for before or after product iondetermination.

From an alternative perspective, the present invention can also bedirected to a method of using quadrupole ion trapping mass spectrometryfor multiplexed determination of multiple parent/product ionrelationships. Such a method comprises providing a quadrupole ion trapmass spectrometer; providing at least two mass spectra, each spectrumcomprising product ions of a plurality of parent ions, with eachspectrum generated at a different rf voltage (or, at a different low m/zcut off value); calculating a fractional change in ion abundance (or,ion intensity) for each parent ion upon change in product ionaccumulation; and determining product ions having a change in abundance,from one spectrum to another, according to the calculated fractionalchange for each parent ion.

The parent ion spectrum can be derived from multiple, two or more,analytes or compounds of interest, simultaneously, as may be provided byway of a compound mixture. Embodiments of this inventive methodology canbe used in conjunction for the structural analysis of multiple or amixture of peptide compounds; however, without limitation, a range ofnon-peptide analytes are also contemplated as would be understood bythose skilled in the art made aware of this invention. While certainembodiments are described in conjunction with first and second production spectra, one or more additional spectra can be utilized as describedherein, depending upon the number of compounds in a mixture, parent ionsanalyzed and/or accuracy desired. The mass spectrometric methods of thisinvention can be used alone or in conjunction with one or morestructural or separatory techniques, including but not limited to highpressure liquid chromatography.

In part, the present invention can also comprise a method of usingGaussian distribution to assess a product ion mass spectrum. Such amethod comprises providing an m/z parent ion accumulation exhibiting aGaussian distribution as a function of ion detection conditions (e.g.,without limitation, applied voltages), the parent ion having a detectioncondition (optimal in certain embodiments), with the distributionproviding a center value, c, and a width value, w, for eachaccumulation, each such distribution generated at an ion kinetic energy;determining a fractional change in parent ion accumulation according tothe relationship(s) described, herein; and applying the fractionalchange to a first product ion spectrum to assess a second product ionspectrum, to associate product ion spectra with a corresponding parention. While detection and/or trapping conditions associated with thisinvention can be described in terms of rf voltages, it is understood inthe art, as illustrated in several examples, that corresponding valuesfor low m/z cut offs (LMCO) can be used as provided directly via thespectrometric instrumentation used. Likewise, equation 1 or a variationof the relationship provided herein, using LMCO values in the mannershown, can be used with comparable effect. As discussed elsewhereherein, ion kinetic energy can be varied to affect ion accumulation.

Regardless, alone, in conjunction or coordinated with, or as an adjunctto tandem mass spectrometry or a multiplexed variation thereof, thisinvention can also be directed to a method of using ion kinetic energyto affect ion trapping conditions in a quadrupole ion trapping massspectrometer. Such a method can comprise varying the kinetic energy ofan m/z ion generated by such a spectrometer, such variation at leastpartially sufficient to change trapping conditions or the range of anysuch condition with respect to the trapping of an m/z ion. Variation inion kinetic energy can be implemented by adjustment or change of acorresponding experimental variable, depending upon the particularspectrometer employed. In certain embodiments, ion kinetic energy can bevaried at least in part with variation of the octopole dc voltage of thespectrometer. In various other embodiments, corresponding changes oradjustments to instrumental hardware, can be used to reduce and/orsubstantially minimize mass bias associated with external ion injectioninto such a spectrometer, such changes/adjustments as would beunderstood by those skilled in the art made aware of this invention.

Likewise, in part, the present invention can be directed to a method oftrapping a plurality of m/z ions in a quadrupole ion trapping massspectrometer. Such a method can comprise accumulating a plurality of m/zions, each such accumulation exhibiting a Gaussian distribution as afunction of voltages applied to the quadrupole ion trap, with each suchm/z ion having a kinetic energy; and varying the ionic kinetic energy,such a variation at least partially sufficient to shift the center of atleast one ion distribution toward the center of another iondistribution. In certain embodiments, accumulation can be a function ofLMCO, and variation can increase the LMCO range for trapping at leastone m/z ion. In certain other embodiments, together with a shift ofdistribution center, such a variation can broaden such a distributionover an increased range of LMCO values. In certain such embodiments,such a range can be increased sufficient to trap two or more such m/zions. Accordingly, such a result can reflect multiple ions over a widem/z range efficiently trapped at a single rf trapping amplitude, to moreaccurately represent the component species present in an analyte sample.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

More particularly, to illustrate several non-limiting embodiments, afteran initial or first product ion spectrum (i.e., primary spectrum) istaken for each of the parent ions simultaneously, another or secondproduct ion spectrum (i.e., secondary spectrum) is acquired with theabundances corresponding to each parent ion changed by a known amount.By doing this the product ions are encoded for by the abundance changeof their parent ion. For example, in FIG. 1, four parent ions from theinitial mass spectrum (FIG. 1A) are isolated (FIG. 1B) andsimultaneously dissociated to give a primary product ion spectrum (FIG.1C). Then the same parent ions (FIG. 1B) are simultaneously dissociatedto obtain a secondary product ion spectrum (FIG. 2B) after changingtheir abundances. The product ions of each parent ion can then bedetermined by observing their resulting abundance change, which shouldbe correlated with the abundance change of their associated parent ion.

FIG. 2A shows the associated abundance change of each parent ion. Aninitial assumption is that the likelihood that two different parent ionswill dissociate to the same product ion m/z is very low. This assumptionallows one to overcome an obvious conundrum, which can be expressed mostclearly by drawing an analogy to mathematically solving for a set ofunknowns. Usually to solve for n unknowns one needs n equations.However, with this methodology, product ion spectra are solved for 4parent ions, using only 2 spectral acquisitions in conjunction with aconstraint on the possible values for the unknowns. (Note: the spectrashown in FIGS. 1B and 2A are hypothetical and for illustration purposesonly.) The approach described above is not limited to 2-4 parent ionsbut is applicable, without limitation, to mass spectra of more complexsystems. A timesaving feature of this approach is that structuralinformation can be gathered from multiple, n, parent ions in as few as 2product ion spectra. Of course, as the number of parent ions increasesthe likelihood that more than one parent ion will dissociate to productions of the same m/z will increase. To address this potentiality, athird product ion spectrum (or tertiary spectrum) can be acquired inwhich the parent ion abundances are changed to another fraction/multipleof their initial abundances. Then, for any given product ion, simplematrix algebra could be used to deconvolute the data.

Changing the parent ion abundances in a known way from one product ionspectrum to the next provides and enables the intensity encoding schemefor the multiplexed MS/MS of this invention. Change in the parent ionabundance can be accomplished by accumulating the parent ions in theprimary spectrum and the secondary spectrum under different trappingconditions. The rf voltage applied to the ring electrode during ionaccumulation can be changed from one value to another in going from theprimary spectrum to the secondary spectrum. The resulting changes inparent ion abundances reflect a feature of ion accumulation in theQITMS: the efficiency of ion accumulation changes in a mass-dependentfashion as the rf voltage is changed. This phenomenon is illustrated inFIG. 3. As the rf voltage is increased, ions of higher m/z are moreefficiently accumulated. From FIG. 3 it is important to note two things.First, the change in ion abundance as a function of rf voltage exhibitsa Gaussian distribution (FIG. 3A). Second, for different m/z ions thereexists an optimum rf voltage at which the ions are most efficientlyaccumulated. The optimum rf voltage clearly increases as the m/z of theion increases (FIG. 3B).

Because Gaussian distributions are well-understood mathematicalfunctions, the abundance changes of any m/z ratios of interest can bepredicted upon changing the rf accumulation voltage as long as thecenter and the width (at ½ height) of the Gaussians are known. For thepeptide ions, for example, the relationship between the center of theGaussian distribution and m/z is linear (FIG. 3B), which has beenobserved previously in the art. Furthermore, the widths (at ½ height) ofthe Gaussian peaks seem to be relatively constant at a value of 41±3 V.Such a value may vary from instrument to instrument and may be somewhatdependent upon operating conditions. Accordingly, an instrument orapparatus used in conjunction with one of the present methods may becalibrated. Nonetheless, typical values can be of the sort describedherein.

Referring back to the data in FIGS. 1C and 2B, the parent ion abundancechanges and thus the product ion abundance changes, which allow theappropriate parent/product ion relationships to be established, can bepredicted by determining the Gaussian distributions for each ion. TheGaussian center and width are used to determine this distribution, and anormalization factor is used to ensure a valid comparison of ionabundances between the primary and secondary spectra. The correspondingcenter for a particular m/z parent ion can be determined using arelationship illustrated in FIG. 3B, the width is 41 V, and anormalization factor can be applied. The fractional change in abundance(F₁₋₂) in going from the primary (i.e., first product ion) spectrum(FIG. 1C) to the secondary (i.e., second product ion) spectrum (FIG. 2B)can then be determined by applying the Gaussian center and width toequation 1, where rf1 and rf2 are the rf accumulation voltages used forthe primary and secondary spectra, c is the center of the Gaussian, andw is the Gaussian width. Using equation 1 the expected fractional$\begin{matrix}{F_{1 - 2} = \frac{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 2} - c})}^{2}}{2\quad w^{2}})}}{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 1} - c})}^{2}}{2\quad w^{2}})}}} & (1)\end{matrix}$change in ion abundance for the parent ion at m/z 524, for example, is0.41. The product ions whose abundances change by approximately thisamount in the secondary spectrum (FIG. 2B) are the ions at m/z 784(0.40), 669 (0.46), 647 (0.45), 466 (0.38), and 263 (0.50). Such ionabundance changes allow these product ions to be associated with theparent ion at m/z 524. This process can be repeated for each of theparent ions in order to determine all the appropriate parent/product ionrelationships. In this way the multiplexed MS/MS data can bedeconvoluted to determine the individual product ion spectra for each ofthe five parent ions used in this illustration.

The product ion spectra for 4 parent ions can be acquired in 50% of thetime it would have taken to acquire the individual product ion spectrausing serial MS/MS (i.e., 2 vs. 4 spectra), a significant increase inefficiency. An associated increase in throughput allows, for instance,structural interrogation of 4 parent ions co-eluting from ahigh-performance liquid chromatographic (HPLC) run. Whereas by serialMS/MS, structural information for only 2 of these parent ions might beattainable. Multiplexed MS/MS on a greater number of parent ions leadsto an even greater relative reduction in analysis time.

Further, this invention represents an improvement over existing QITMStechnology by way of possible structural analyses of complex compoundmixtures. Often liquid chromatography (LC) is combined with MS tofacilitate the study of mixtures. LC is used to separate a mixture intoits components, and then the separated components are introduced intothe mass spectrometer one at a time for structural analysis. In caseswhere short analysis times are necessary, LC runs are oftensubstantially shortened. As a consequence more components elute into themass spectrometer simultaneously. If, for example, multiple compoundselute into the mass spectrometer during a 4 sec period, serial MS/MS onthe QITMS would likely only allow two parent ions to be structurallyanalyzed. In contrast, the present multiplexed MS/MS technique allowsall of the co-eluting parent ions to be structurally analyzed, whichwould maximize the available information.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the methods of the present invention. Incomparison with the prior art, the present methods provide results anddata which are surprising, unexpected and contrary thereto. While theutility of this invention is illustrated through the use of severalpeptide or protein mixtures, it will be understood by those skilled inthe art that comparable results are obtainable with various othercompounds or number thereof, as commensurate with the scope of thisinvention.

Likewise, the methods of this invention can be practiced withoutlimitation as to any one QITMS apparatus, component configuration orassociated software. For instance, while isolated parent ions can bedescribed as resonantly excited via waveforms corresponding to theiroscillation frequencies to induce energetic collisions with a backgroundgas (e.g., helium) and dissociation into product ions, those skilled inthe art would understand other methods to achieve dissociation intoproduct ions in a quadrupole ion trap. For instance, with appropriatecomponent modification, photons from infrared lasers can be used todissociate parent ions. Various other QITMS apparatus and componentconfigurations, together with related software, programs and associatedhardware, are commercially-available and known in the art. For example,reference is made to the aforementioned, incorporated '884 patent, inparticular FIGS. 1-2 thereof, such apparatus and components as can beused herewith, through straight-forward modifications thereof, as wouldbe understood by those skilled in the art made aware of this invention.

Example 1

As discussed above, a commonly considered disadvantage of quadrupole iontrap mass spectrometers is the limited and variable m/z range over whichions can be efficiently accumulated from external ion sources. This factis illustrated in FIG. 4, which shows the mass spectra of the proteincytochrome c at two different rf accumulation voltages. In FIG. 4Acytochrome c charge states ranging from +15 to +20 are apparent when therf voltage applied to the ring electrode during ion accumulation is suchthat the low m/z cut off (LMCO) is 41 Da, which corresponds to an rfvoltage of approximately 257 V_(p-p). When the rf accumulation voltageis increased to 320 V_(p-p) (LMCO=51 Da) during ion accumulation, thecytochrome c mass spectrum (FIG. 4B) clearly changes so that chargestates between +13 and +18 are apparent. If the normalized ion intensityof the +15 charge state is plotted over a range of LMCO values, it isclear that a Gaussian distribution represents the relationship betweenion intensity and LMCO during ion accumulation (FIG. 5A). Furthermore,plotting the normalized ion intensities of other charge states resultsin Gaussian distributions that differ by center of the distribution, andthe distribution center increases as the mass-to-charge ratio (m/z) ofthe ion increases.

Example 2

A series of 16 peptide ions were studied to observe the relationshipbetween ion intensity and the LMCO during ion accumulation. Just as inthe preceding example, the distribution of ion intensity as a functionof LMCO is also Gaussian in nature. Moreover, there is a linearrelationship between the optimum LMCO during accumulation and peptideion m/z, as illustrated in FIG. 6. The slope of this linear relationshipdiffers for peptide ions with a +1 charge state as compared to peptideions with a +2 charge state. For the +1 peptide ions, the equation forthe line in FIG. 3 is y=0.024x+33.2, while for the +2 peptide ions theequation is y=0.045x+21.7. The width (at 50%) of the Gaussiandistribution does not have any apparent relationship with peptide ionm/z as evidenced by FIG. 7. Within experimental error, the Gaussianwidths (at 50%) appear to be about 8 Da. Both the Gaussian centers andwidths are somewhat dependent upon the parameters used to transport theions from the electrospray ion source to the quadrupole ion trap. Theseparameters were held constant during all the studies described, below.Optimal application of this intensity encoding scheme can depend uponinstrumental calibration to determine the appropriate Gaussian widthsand the equations that relate the Gaussian centers to m/z values.

As demonstrated above, the reproducible Gaussian relationship betweenion intensity and LMCO during ion accumulation allows the development ofan intensity encoding scheme for multiplexed MS/MS. Application of thisintensity encoding scheme is illustrated in FIG. 8. If a given set ofparent ions is accumulated at one LMCO (or rf voltage, V) during theacquisition of the primary spectrum and then at a different LMCO (or rfvoltage, V) during the acquisition of the secondary spectrum, the ions'changes in intensities can be predicted based upon the known Gaussiandistributions. Equation 1 is used to calculate this fractional intensitychange. In a variation of the relationship given by equation 1 LMCO(1°)(i.e., corresponding to rf1) is the low m/z cut off used for ionaccumulation in a first or primary spectrum, LMCO(2°) (i.e.,corresponding to rf2) is the low m/z cut off used for ion accumulationin a second or secondary spectrum, c is the center of the Gaussiandistribution, which is m/z dependent and can be determined from lines inFIG. 6, and w is the width (at 50%) of the Gaussian distribution, whichis equal to ˜8 Da for each parent ion. $\begin{matrix}{F_{{1{^\circ}} - {2{^\circ}}} = \frac{{\mathbb{e}}^{(\frac{- {\lbrack{{{LMCO}{({1{^\circ}})}} - c}\rbrack}^{2}}{2\quad w^{2}})}}{{\mathbb{e}}^{(\frac{- {\lbrack{{{LMCO}{({2{^\circ}})}} - c}\rbrack}^{2}}{2\quad w^{2}})}}} & (2)\end{matrix}$

Example 3

The data of Examples 3a-3e were acquired using a Bruker Esquire-LCquadrupole ion trap mass spectrometer. The following Esquire parameterswere used for generation of the spectra. All parameters are subject tochange depending on the compounds analyzed and correspondingexperimental procedure. Some parameters vary slightly from day-to-dayand are chosen to optimize a particular instrument response. Theseparameters are noted with and asterisk (*). The parameters noted with aplus sign (+) are optimally kept as constant as possible, as they appearto affect Gaussian width and center values, more so than others.*Capillary −3000 V *End Plate Offset −500 V *Nebulizer 5 psi *Dry gas 3L/min *Dry temp. 300 C. +Skimmer 1 30 V +Skimmer 2 10 V +Capillary ExitOffset 80 V +Capillary Exit 110 V +Octopole 2.70 V +Octopole Delta 2.50V *Octopole RF 175 V(p —p) *Lens 1 −5 V *Lens 2 −60 V Multiplier −1370 VDynode −7 kV Scan Delay 0.50 ms Scan 50 to 1650* Isolation Delay 0 msFragmentation Delay 10 ms Esquire Isolation mass 318 - approx qz 0.8+SWIFT dissociation time 65 ms +SWIFT isolation time 65 ms +SWIFTdissociation amplitude  2 V(p—p) for all ions +SWIFT isolation voltage10 V(p—p) for all ions +SWIFT dissociation width 10 m/z units centeredaround ion of interest +SWIFT isolation width 10 m/z units centeredaround ion of interest

SWIFT is an algorithm used to help isolate and dissociate parent ions.The SWIFT waveforms were calculated using a program written in LabView6.0 and then downloaded to a Wavetek, Model 39 arbitrary waveformgenerator. A Stanford Research Systems, INC., Model DG535 digital pulsegenerator was then used to apply the SWIFT isolation and dissociationwaveforms with the appropriate timing to the entrance endcap electrodeof the Bruker Esquire-LC. Nonetheless, in conjunction with thisinvention, various other methods can be used to facilitate ion isolationand dissociation. Likewise, as would be understood by those skilled inthe art, other hardware components can be used to generate appropriatewaveforms.

The collected mass spectral data was exported from Bruker's DataAnalysis program into Microcal Origin 6.0 for further data processing.Origin 6.0 was also used to fit the Gaussian peaks and calculate thefractional changes from one point on the Gaussian distribution toanother.

Example 3a

The intensity encoding aspect of this invention was applied to acollection of 5 peptide ions, as illustrated in the following. A mixtureof 5 peptide, including TRH-Gly, angiotensinogen, leucine enkephalin,β-casomorphin, and leucokinin, were analyzed by a multiplexed MS/MSapproach of the sort described herein. FIG. 9A displays the massspectrum of these 5 peptide accumulated at a LMCO of 48 Da (rfvoltage=301 V_(p-p)), and FIG. 9B shows the mass spectrum of these 5singly-charged peptide ions after isolatation using the stored-waveforminverse Fourier transform (SWIFT) method. The [M+H]⁺ ions of thesepeptides appear at m/z 421, 482, 556, 659, and 783 for TRH-Gly,angiotensinogen, leucine enkephalin, β-casomorphin, and leucokinin,respectively. Simultaneous dissociation of these 5 peptide ions using asecond SWIFT waveform results in a first product ion spectrum shown inFIG. 10.

Example 3b

The same 5 peptide ions are then accumulated at a different LMCO value,with their intensities changed as described above. FIG. 11 shows themass spectrum of the 5 SWIFT isolated peptide after ion accumulation ata LMCO of 58 Da (rf voltage=364 V_(p-p)). Comparing the spectrum withFIG. 9B, it is evident that the parent ion intensities have changed. Theexpected parent ion intensity changes can be calculated using equation1, and Table 1 provides a comparison of the experimentally observed andpredicted intensity changes after accumulating the ions at this new LMCOvalue. Table 1 shows that with the exception of the peptide TRH-Gly theobserved and calculated intensity changes are very close. (A discussionabout the deviation for TRH-Gly is provided in example 3e, below).Simultaneous SWIFT dissociation of these 5 peptide ions, accumulated atthe new LMCO value, results in a second product ion spectrum shown inFIG. 12. In actual practice, the spectrum shown in FIG. 11 would notneed to be acquired because the intensity changes are predictable,according to the relationship of Equation 1. (In other words, FIG. 11illustrates the point that the parent ion intensities change in apredictable manner.) TABLE 1 Experimentally Observed and CalculatedIntensity Changes for the 5 Peptide Ions. Observed Intensity CalculatedIntensity Peptide Ion Ratio (1°/2°)^(a) Ratio (1°/2°)^(b) Leucokinin(m/z 783) 1.2 1.2 β-casomorphin (m/z 659) 2.1 1.9 Leucine enkephalin(m/z 2.4 2.7 556) Angiotensinogen (m/z 482) 3.2 3.7 TRH-Gly (m/z 421)1.9 4.5^(a)Determined by dividing the ion intensity from the spectrum in FIG.9B by the ion intensity in FIG. 11.^(b)Determined using the LMCO variation of equation 1

Example 3c

A comparison of the first product ion spectrum shown in FIG. 10 with asecond product ion spectrum in FIG. 12 indicates that the intensities ofmany of the product ions have changed, although a simple visualinspection of these two spectra may not, in all instances, clearlyindicate the intensity changes. A more convenient means of determiningthe intensity changes and thus identifying the parent ion from whicheach product ion arises is to use a ratio spectrum. If the primary(product ion) spectrum in FIG. 10 is divided by the secondary (production) spectrum in FIG. 12, a ratio spectrum is obtained (FIG. 13). FIG.13 was obtained by first subtracting out any ions with intensities below150, which removes any noise that would ultimately be amplified in theratio spectrum while assuming no useful information is deleted. From theratio spectrum in FIG. 13, linking the parent and product ions can beaccomplished by identifying the product ion m/z values that appear atthe ratios shown in Table 1. For instance, FIG. 14 shows an expandedregion of FIG. 13 from m/z 450 to 625. In this region 13 differentproduct ions are present. The m/z and intensity ratios of these productions are listed in Table 2 along with their parent ions, which weredetermined by comparing the product ions' intensity ratios with theexpected parent ion ratios shown in Table 1. With the exception of m/z493 and 526, the intensity encoding scheme allowed all the product ionsin this range to be correctly associated with their appropriate parention. The product ions at m/z 493 and 526 both have intensity ratios ofabout 1.8, while their parent ions, leucine enkephalin and leucokinin,were encoded at 2.4 and 1.2, respectively. Close inspection of FIGS. 10and 12 indicate that m/z 493 has intensities of 274 and 150,respectively, while m/z 526 has intensities of 288 and 155,respectively. The later intensities, each in the corresponding secondaryspectrum, are both very close to the value of 150 chosen for noiseremoval, showing the foregoing assumption can be adjusted as needed.TABLE 2 Product Ions Between m/z 450 and 625 in the Ratio Spectrum (FIG.14) and Their m/z Ratios, Intensity Ratios, and Identified Parent Ions.Product Ion Identified Parent Ion Product Ion (m/z) Intensity Ratio(1°/2°) (Parent Ion Intensity Ratio)^(a) 465 2.4 Leucine enkephalin(2.4) 487 1.3 Leucokinin (1.2) 493 2.0 — 495 2.2 β-casomorphin (2.1) 5002.2 β-casomorphin (2.1) 505 1.2 Leucokinin (1.2) 526 1.8 — 538 2.6Leucine enkephalin (2.4) 597 2.2 β-casomorphin (2.1) 614 2.1β-casomorphin (2.1)^(a)Parent ion intensity ratio from Table 1

Example 3d

Another means by which this data can be displayed is to show theauthentic MS/MS spectrum of one of the parent ions with an indication ofthe product ions identified during the multiplexed MS/MS analysis. FIG.15 shows the product ion spectrum for leucokinin. The peaks with anasterisk (*) above them are the product ions that were correctlyidentified during the multiplexed MS/MS experiment. The peaks with theplus sign (+) above them are product ions that were not identifiedduring the multiplexed MS/MS experiment because these ions have m/zvalues close to those of other parent ions and thus their detection isprecluded by the SWIFT waveforms used to dissociate these parent ions.Product ions at m/z 422, which is close to the m/z of TRH-Gly (m/z 421),and m/z 655, which is close to the m/z of β-casomorphin (m/z 659) arethe two ions that fall into this category. The peak with a question mark(?) above it corresponds to a product ion that was present in the ratiospectrum, but its intensity ratio was deemed to be too far from theexpected intensity ratio for leucokinin (i.e. 1.2). This ion at m/z 526was found to have an intensity ratio of 1.8. Again, as noted above, amiscoding of this ion is likely due to its low intensity, which is closeto the noise level. The other peaks in FIG. 15, which were notidentified, likely were removed when the “noise” was subtracted toobtain the ratio spectrum in FIG. 13. As noted above some usefulinformation is lost by removing the noise.

Example 3e

As observed in Table 1, the peptide TRH-Gly (m/z 421) was not optimallyencoded. The observed encoding of m/z 421 can be understood by observingFIG. 16. The ion intensity vs. LMCO plot for this ion shows some“tailing” of the Gaussian distribution at higher LMCO values. Thistailing occurs to some degree for all peptide ions, but it only has adetrimental effect on predicting the intensity change if the LMCO valuesused to acquire both the primary and secondary spectra occur in this“tailing” region. The lines in FIG. 16 show the LMCO values at which theprimary and secondary spectra were acquired for FIGS. 9 through 12. Afirst assumption useful in conjunction with this methodology is that thedistribution of ion intensities as a function of LMCO is Gaussian.However, the distribution may indeed be a modified Gaussiandistribution. The effects of ion injection parameters on thisdistribution may, for a particular ion, suggest an adjustment of primaryand secondary rf voltages to avoid spectra acquisition in such a tailingregion.

Example 4

To promote a better fundamental understanding of why a Gaussianrelationship exists between ion abundance and the LMCO used during ionaccumulation, effort has been made to understand how to control/changethese Gaussian distributions for better performance. From this work, itwas identified that the distribution of ion kinetic energies as ionsenter the quadrupole ion trap is a factor easily controllable from anexperimental standpoint if one desires to control/change the observedGaussian distributions.

An experimental variable on the particular quadrupole ion trap massspectrometer used was found to easily vary the distribution of ionkinetic energies. For any quadrupole ion trap mass spectrometer in whichions are generated externally, a series of electrodes with appliedvoltages are used to direct ions into the mass analyzer. Theseelectrodes and the associated voltages applied to them ultimatelycontrol the distribution of ion kinetic energies. On the BrukerEsquire-LC quadrupole ion trap mass spectrometer described above, it wasobserved that variation of the dc offset on octopole 1 provides goodexperimental control of the ion kinetic energy distribution. By varyingthe dc potential applied to this lens element, ion abundance as afunction of the LMCO can be significantly changed (see FIG. 17). Becausethe present methods relate to distributions like those shown in FIG. 17,understanding how to control these distributions can be used to increasethe overall flexibility of this spectrometric approach.

As shown above, with reference to Examples 1-4, multiplexed MS/MS can beused for a collection of n (e.g., in this example 3, n=5) peptide ionsin which only two product ion spectra are necessary to obtain theappropriate relationships between parent and product ions. This is anadvantage over serial MS/MS in which five product ion spectra would berequired to obtain the same information. The multiplexed MS/MS approachof this invention encodes the parent ions according to their intensity.This intensity encoding can be accomplished by recognizing that in aquadrupole ion trap mass spectrometer the intensity of a given parention displays a Gaussian distribution with respect to the rf voltageapplied to the ring electrode during ion accumulation. The centers ofthese Gaussian distributions increase in a linear fashion as the m/zratios of the parent ions increase. Furthermore, the widths (at 50%) ofthese distributions are relatively constant and independent of m/z. Bychanging the rf accumulation voltage from one spectrum to the next,parent ions (and thus their resulting product ions) can be encoded in apredictable manner. The appropriate parent/product ion relationships canthen be determined by comparing the intensity changes of the productions with the expected intensity changes of the parent ions.

Example 5

The impact of varying ion kinetic energy during ion injection into theQITMS can be demonstrated by determining the trapping efficiency of ionsas a function of rf trapping amplitude. FIG. 18 demonstrates iontrapping efficiency as a function of rf voltage under normal operatingconditions (i.e. no modulation of ion kinetic energy). In this figure rfvoltage is given as the LMCO, which as discussed above, is aninstrumental parameter directly related to the actual rf voltage. Use ofLMCO, instead of rf voltage, generalizes results for direct comparisonwith other QITMS. FIG. 18 is instructive: first, ion trapping efficiencydepends upon the LMCO used during ion accumulation. Second, trappingefficiency for a given ion is optimal at a single LMCO value. Third,depending upon the ion's m/z ratio, the optimum LMCO value is different.Fourth, under normal operating conditions, it is impossible to choose asingle rf voltage that allows optimal trapping of ions with differentm/z ratios; for example, conditions that lead to optimal trapping of m/z1060 lead to very inefficient trapping of m/z 556 and visa versa. If onechanges the ions' kinetic energies, the centers and widths of thedistributions shown in FIG. 18 will change. The ions illustrated arenon-limiting and are representative of mixtures of analytes (e.g.,peptides) of the sort which can be analyzed as described herein.

Example 6

FIG. 19 demonstrates the change in distribution centers and widths as afunction of the octopole dc voltage for an ion at m/z 556. The octopoledc voltage is the main experimental variable of the Bruker Esquire-LC(as discussed above) that controls ion kinetic energy as ions approachthe ion trap. (Other QITMS products, as known in the art, can employchange of different experimental variables, such variables limited onlyby function to vary ion kinetic energy.) FIG. 18 clearly shows that boththe distribution centers and widths change when ion kinetic energy ischanged.

Example 7

FIG. 20 shows trapping efficiency as a function of LMCO for m/z 556 and1060 when the ions enter the trap with higher kinetic energies than inFIG. 18. In comparing FIGS. 18 and 20, it becomes evident that if ionkinetic energies are modulated during ion accumulation a single rftrapping voltage might be used to approach equal trapping efficiency fora wide range of ions.

Example 8

FIG. 21 demonstrates the trapping efficiency as a function of LMCO foran ion at m/z 556 at 8 different kinetic energies. In comparison to FIG.18, this figure demonstrates improvement in trapping efficiency over avery wide range of LMCO values (i.e. rf trapping voltages). By varyingion kinetic energy during ion accumulation, m/z 556 would be efficientlytrapped at LMCO values that range from 30 to 110. Under the operatingconditions (i.e., normal) of Example 5, this ion would only beefficiently trapped at LMCO values between 40 and 55. This improvementis possible for other ions as well, and when a collection of ions with awide range of m/z ratios are simultaneously analyzed, all the ions canbe trapped with near equal efficiency at one rf trapping amplitude.

As demonstrated above, with reference to Examples 5-8, by varying (ormodulating) ions kinetic energies during transport from the ion sourceto the mass analyzer, the mass bias associated with ion accumulation ina quadrupole ion trap mass spectrometer can be substantially minimized.The nature (i.e. sine wave, square wave, etc.) and frequency of themodulation can conceivably take many forms, but an optimum frequency canexist for a given working condition.

1. An ion trap mass spectrometric method for resolving a mixture ofcompounds, said method comprising: providing first spectra comprising aspectrum of a plurality of compound parent ions, and a spectrum of atleast one product ion of each said parent ion, said first spectraacquired under a first set of ion trapping conditions; providing secondspectra comprising a spectrum of a plurality of compound parent ions,and a spectrum of at least one product ion of each said parent ion, saidsecond spectra acquired under a second set of ion trapping conditions;determining the fractional change in each parent ion accumulated oversaid first and second spectra; and applying said parent ion fractionalchange to at least one of said product ions in said first and secondproduct ion spectra, to determine a product for each said parent ion. 2.The method of claim 1 wherein said ion trapping conditions are selectedfrom at least one of rf voltage applied to a ring electrode during saidaccumulation, and a predetermined low m/z cut off.
 3. The method ofclaim 2 wherein said spectra are acquired over different applied rfvoltages, and said fractional change, F₁₋₂, is expressed by$\begin{matrix}{F_{1 - 2} = \frac{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 2} - c})}^{2}}{2\quad w^{2}})}}{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 1} - c})}^{2}}{2\quad w^{2}})}}} & (1)\end{matrix}$ wherein e is the natural log base, rf1 is a first appliedvoltage and rf2 is a second applied voltage, c is the center of aGaussian distribution of said parent ion accumulation, and w is thewidth at one-half the height of said Gaussian distribution.
 4. Themethod of claim 1 wherein said parent ion accumulation is measured byone of ion abundance and ion intensity.
 5. The method of claim 4comprising varying ion kinetic energy, said variation at least partiallysufficient to enhance said ion accumulation.
 6. The method of claim 1comprising provision of third spectra comprising a spectrum of aplurality of compound parent ions, and a spectrum of at least oneproduct ion of each said parent ion, said third spectra acquired under athird set of ion trapping conditions.
 7. The method of claim 1comprising a liquid chromatographic separation selected from prior tosaid product ion determination and after said product ion determination.8. A method of using quadrupole ion trap mass spectrometry formultiplexed determination of multiple parent/product ion relationships,said method comprising: providing a quadrupole ion trapping massspectrometer; providing at least two mass spectra, each said spectrumcomprising product ions of a plurality of parent ions, each saidspectrum generated at an ion kinetic energy during ion introduction intosaid spectrometer, each said spectrum generated at a different rfvoltage; calculating a fractional change in ion abundance for each saidparent ion; and determining product ions having a change in abundance,over each said spectrum, corresponding to said calculated fractionalchange for each said parent ion.
 9. The method of claim 8 wherein saidfractional change, F₁₋₂, is expressed by $\begin{matrix}{F_{1 - 2} = \frac{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 2} - c})}^{2}}{2\quad w^{2}})}}{{\mathbb{e}}^{(\frac{- {({{{rf}\quad 1} - c})}^{2}}{2\quad w^{2}})}}} & (1)\end{matrix}$ wherein e is the natural log base, rf1 is a first appliedvoltage and rf2 is a second applied voltage, c is the center of aGaussian distribution of said parent ion accumulation, and w is thewidth at one-half the height of said Gaussian distribution.
 10. Themethod of claim 8 comprising generation of two mass spectra.
 11. Themethod of claim 10 wherein said determination comprising acquisition ofa ratio spectrum comparing the quotient of said spectra with saidfractional change in parent ion abundance.
 12. The method of claim 8further comprising varying ion kinetic energy to enhance ion abundance.13. A method of using quadrupole ion trapping mass spectrometry formultiplexed determination of a peptide mixture, said method comprising:providing a quadrupole ion trapping mass spectrometer; providing atleast two mass spectra, each spectrum comprising product ions of aplurality of peptide parent ions, each said spectrum generated underdifferent ion trapping conditions, said conditions selected from one ofrf voltage and a predetermined low m/z cut off; calculating a fractionalchange in ion accumulation for each said peptide parent ion; anddetermining product ions having an accumulation change, over saidspectra, corresponding to said calculated fractional change for eachsaid peptide parent ion.
 14. The method of claim 13 wherein saidfractional change, F₁₋₂, is expressed by$F_{{1{^\circ}} - {2{^\circ}}} = \frac{{\mathbb{e}}^{(\frac{- {\lbrack{{{LMCO}{({1{^\circ}})}} - c}\rbrack}^{2}}{2\quad w^{2}})}}{{\mathbb{e}}^{(\frac{- {\lbrack{{{LMCO}{({2{^\circ}})}} - c}\rbrack}^{2}}{2\quad w^{2}})}}$wherein e is the natural log base, LMCO(1°) is a first low m/z cut offand LMCO(2°) is a second low m/z cut off, c is the center of a Gaussiandistribution, and w is the width at one-half the height of said Gaussiandistribution.
 15. The method of claim 14 wherein said peptide parent ionaccumulation is measured by one of ion abundance and ion intensity. 16.The method of claim 15 comprising varying ion kinetic energy, saidvariation at least partially sufficient to enhance said ionaccumulation.
 17. The method of claim 14 wherein said peptide parent ionaccumulation is measured by ion intensity.
 18. The method of claim 17comprising generation of two spectra.
 19. The method of claim 18 whereinsaid determination comprises acquisition of a ratio spectrum comparingthe quotient of said spectra with said fractional change in peptideparent ion intensity.
 20. The method of claim 13 used with a proteomicassessment.
 21. A method of using Gaussian distribution to assessproduct ion mass spectra from a quadrupole ion trapping massspectrometer, said method comprising: providing an m/z parent ionaccumulation exhibiting a Gaussian distribution as a function ofvoltages applied to the quadrupole ion trap, with said distributionproviding a center value, c, and a width value, w, for each saidaccumulation, each said distribution generated at an ion kinetic energy;determining a fractional change in parent ion accumulation; and applyingsaid fractional change to a first product ion spectrum to assess asecond product ion spectrum, to associate said product ion spectra witheach said parent ion.
 22. The method of claim 21 wherein said parent ionaccumulation is measured by one of ion abundance and ion intensity. 23.The method of claim 21 wherein said voltages are applied to a ringelectrode during said accumulation.
 24. The method of claim 21comprising varying said ion kinetic energy, said variation at leastpartially sufficient to shift the center of at least one saiddistribution toward the center of another said distribution, saidvariation enhancing said ion accumulation.
 25. A method of using ionkinetic energy to affect ion trapping conditions in a quadrupole iontrapping mass spectrometer, said method comprising varying the kineticenergy of an m/z ion generated by a quadurpole ion trapping massspectrometer, said variation at least partially sufficient to change thecondition range for trapping said ion.
 26. The method of claim 25wherein said ion trapping conditions are selected from at least one ofrf voltage applied to a ring electrode during said accumulation, and apredetermined low m/z cut off.
 27. The method of claim 26 wherein saidcondition is a predetermined low m/z cut off.
 28. The method of claim 27wherein said condition change comprises trapping said m/z ion over anincreased range of low m/z cut off values.
 29. The method of claim 28wherein said ion accumulation is measured by one of ion abundance andion intensity.
 30. The method of claim 25 where said ion kinetic energyis varied at least in part with variation of the octopole dc voltage ofsaid mass spectrometer.
 31. The method of claim 25 comprising aplurality of different m/z ions, said ion kinetic energy variation atleast partially sufficient to change the condition range for trapping atleast one of said ions.
 32. The method of claim 31 wherein said iontrapping conditions are selected from at least one of rf voltage appliedto a ring electrode during accumulation, and a predetermined low m/z cutoff.
 33. The method of claim 32 wherein said condition change comprisesan increased range of low m/z cut off values, said increased rangesufficient for trapping said one ion and at least one said different m/zion.
 34. The method of claim 25 wherein said ion kinetic energy isvaried with variation of the octopole dc voltage of said massspectrometer.
 35. A method of trapping a plurality of m/z ions in aquadrupole ion trapping mass spectrometer, said method comprising:accumulating a plurality of m/z ions, each said accumulation exhibitinga Gaussian distribution as a function of voltages applied to thequadrupole ion trap, each said m/z ion having a kinetic energy; andvarying said ion kinetic energy, said variation at least partiallysufficient to shift the center of at least one said distribution towardthe center of another said distribution.
 36. The method of claim 35wherein said accumulation is a function of a predetermined low m/z cutoff.
 37. The method of claim 36 wherein said variation increases the lowm/z cut off range for trapping at least one of said m/z ion.
 38. Themethod of claim 37 wherein said variation broadens said distribution ofsaid ions over an increased range of low m/z cut off values.
 39. Themethod of claim 38 wherein said low m/z cut off range is increasedsufficient to trap at least two of said m/z ions.
 40. The method ofclaim 35 wherein said ion kinetic energy is varied at least in part withvariation of the octopole dc voltage of said mass spectrometer.